Mutagenesis, Vol. 14, No. 1, 63-66,
January 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Comparison of the aneugenic effect of vinorelbine and vincristine in cultured human lymphocytes
Departamento de Genética, Instituto de Investigaciones Hematológicas `Mariano R.Castex', Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina
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Vinca alkaloids are used clinically against a variety of hematological and solid tumors. These compounds interact with tubulin subunits to prevent microtubule assembly, inducing abnormal chromosome segregation in dividing cells and causing aneuploidy. The vinca alkaloid vincristine sulfate (VCR) and the semisynthetic analog vinorelbine (VRB) were studied by analysis of micronuclei (MN) in cultured human lymphocytes using the cytokinesis block method. Futhermore, fluorescence in situ hybridization with a human alphoid satellite pancentromeric DNA probe was used to detect centromeres in isolated MN of VRB- or VCR-treated lymphocytes. At all the doses tested, both chemicals induced a significant increase in MN frequencies in binucleated (BN) cells (P < 0.001). The maximal effect was reached at a dose of 0.50 µg/ml. At this dose, VRB produced an ~5-fold increase with respect to the control frequency of MN, while with VCR, this frequency increased 10-fold. Both drugs produced a slowing of the cell cycle, causing a decrease in the percentage of BN cells. This effect was lower with VRB. The percentages of centromere-positive MN were 89.79 and 87.60% in VRB- and VCR-treated cultures, respectively (control 27.03%). The high percentage of positive-signals in treated cultures (P < 0.001) indicates that the MN contained whole chromosomes. Our results confirm the aneugenic mode of action of these chemicals, VRB having less genetic effect.
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Introduction |
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Vincristine sulfate (VCR) is a dimeric alkaloid isolated from the periwinkle plant, Catharanthus roseus, which is used for the treatment of several forms of malignancy (Ferguson and Pearson, 1996
). However, the clinical use of this drug is limited, chiefly because it induces sensorimotor neuropathies (Binet et al., 1990). Moreover, many studies have shown that VCR induces a wide spectrum of divisional aberrations which result in mitotic arrest, multipolar spindles, polyploidy and aneuploidy in a variety of in vitro and in vivo test systems (Miller and Adler, 1989; Natarajan et al., 1993; Jackson et al., 1996).
Thus, there has been interest in developing new analogs of vinca alkaloids in order to improve effectiveness and to decrease neurotoxicity in treated patients. Among them, a semisynthetic alkaloid agent, vinorelbine (VRB) has been prepared. Several studies have established that this drug is active in the treatment of different tumours, including non-Hodgkin's lymphoma, Hodgkin's disease, melanoma, head and neck cancer, breast cancer and non-small cell lung cancer. Toxicity has consisted mainly of neutropenia and leukopenia, with minimal non-hematological toxicity (Burris and Fields, 1994; Budman, 1997).
Similar to the other vinca alkaloids, VRB binds to tubulin, inhibiting tubulin polymerization and assembly of mitotic spindle microtubules (Lobert et al., 1996). In addition, this drug inhibits cell proliferation, producing an accumulation of cells in the mitotic phase in cell lines (Pauwels et al., 1995; González Cid et al., 1997a) and in human lymphocytes (González Cid et al., 1997b).
The genetic end point chosen to evaluate aneuploidy was the micronucleus assay in cytokinesis-blocked human lymphocytes. Micronuclei (MN) were derived from either acentric chromosome fragments or whole chromosomes that are not included in the daughter nuclei following nuclear division (Countryman and Heddle, 1976). The micronucleus assay has been greatly improved by the use of cytochalasin B, an inhibitor of cytokinesis, that blocks cultured cells at the end of the first mitotic division, inducing binucleated cells (Fenech and Morley, 1985).
In order to discriminate MN produced by agents causing chromosome breakage (clastogens) from those arising following treatment with agents causing spindle malfunctioning (aneugen), the fluorescent in situ hybridization technique (FISH) with a centromere-specific 
-satellite DNA probe was used (Eastmond and Pinkel, 1990; Farooqi et al., 1993).
The aim of the present study was to evaluate and to compare the effects of the compounds VRB and VCR on human lymphocyte cultures using the micronucleus assay. Futhermore, the content of MN was assessed by the detection of centromeres in isolated MN.
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Materials and methods |
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Chemicals
VCR, vincristine sulfate (CAS no. 2068-78-2), was purchased from Gador (Buenos Aires, Argentina). VRB, 5'-nor-anhydrovinblastine (CAS no. 71486-22-1), was commercially obtained as Navelbine® from Rontag Laboratories (Buenos Aires, Argentina). Colchicine (COL) (CAS no. 64-86-8; Gibco BRL, Grand Island, NY) was included as a positive control compound. All chemicals were dissolved in bidistilled water.
Lymphocyte cultures and MN scoring
Heparinized blood samples were obtained by venipuncture from four healthy non-smoking donors for each compound (three women and one man, aged from 30 to 50 years and free of any known exposure to genotoxic agents). For each culture, a buffy layer leukocyte suspension (0.5 ml) was cultured in 4.5 ml F-10 medium with 15% fetal calf serum (FCS) and 2% phytohemagglutinin (PHA). All cultures were incubated at 37°C for 72 h. After 24 h incubation, cells were treated with 0.05, 0.10, 0.50 or 1.00 µg/ml VRB or VCR. Positive control cultures were treated with COL at a dose of 0.01 µg/ml. For each dose duplicate cultures were prepared. Cytokinesis was blocked with 3 µg/ml cytochalasin B, added 44 h after PHA stimulation. For preservation of the cytoplasm, the cells were processed according to Lee et al. (1994) with some modifications.
Finally, the cells were stained for 10 min in Giemsa (4%) in Sorensen buffer (pH 6.8). Scoring of MN was restricted to binucleated cells (BN) with well-preserved cytoplasm. Where possible, 500 BN cells/dose level were analyzed. The cell cycle kinetics were measured using the percentage of BN cells.
Isolation of MN
A buffy layer leukocyte suspension (0.5 ml) from two healthy women was added to 4.5 ml F-10 medium supplemented with 15% FCS and 2% PHA. After 24 h incubation, cultures were treated with 0.10 µg/ml VRB or VCR. Control (without drug) and positive control (0.01 µg/ml COL) cultures were grown under identical conditions. Cells were harvested after 72 h incubation at 37°C. Isolation of MN was performed following the method of Hayashi et al. (1994). Briefly, the cell pellet was treated with hypotonic solution (0.075 M KCl) for 5 min at room temperature and subsequently methanol:acetic acid (3:1) was added. After 5 min, the cells were centrifuged at 1500 r.p.m. for 15 min and the supernatant was discarded. The pellet was resuspended in 5 ml fresh fixative and filtered through a polycarbonate nuclepore membrane with pores of 2 µm diameter (Millipore, Ireland). After centrifugation, MN were mounted on slides and air dried. The slides were fixed with methanol for 5 min and kept at room temperature until used for FISH.
Fluorescence in situ hybridization (FISH)
The slides were pretreated with 2x SSC (pH 7.0) at 37°C for 30 min and denatured in 70% formamide in 2x SSC (70°C for 2 min), followed by dehydratation in a 70, 90 and 100% ethanol series. The alphoid all human centromere-specific DNA digoxigenin-labeled-probe (Oncor Inc.) was denatured (70°C for 5 min) and applied to the slides under a plastic coverslip. Hybridization was performed at 37°C overnight in a moist hybridization box. After this the slides were washed with 1x SSC (72°C for 5 min) and then for 1–2 min in Tween-20 buffer (0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.2% Tween-20) at room temperature. Detection of the digoxigenin-labeled probe was performed with fluorescein isothiocyanate-labeled anti-digoxigenin under a plastic coverslip for 15 min at 37°C in a moist chamber. Slides were washed three times in Tween-20 buffer for 2 min each. Finally, the preparations were stained with propidium iodide (0.3 µg/ml) in antifade solution. The presence of centromere signals was analyzed in a minimun of 100 MN.
Statistical analysis
The extended 
2 test was used for the assessment of significance of micronucleated binucleated cells (BNMN) over controls. The percentage of BN cells was analyzed using Student's t-test. The 2 test was used to analyze the results obtained with the FISH technique (% MN with positive signals in treated cultures versus % MN positive in control cultures). The dose–response relationships were determined by means of the regression coefficients.
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Results |
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The induction of micronucleated lymphocytes by VRB and VCR is shown in Table I
. Even at the lowest dose (0.05 µg/ml) a significant increase in the MN frequency was observed for both compounds (P < 0.001). The maximal effect was reached at a dose of 0.50 µg/ml. At this dose, VRB induced a frequency of MN of 13.3 ± 3.0 per 1000 BN lymphocytes (control 2.5 ± 0.6). In contrast, VCR produced a frequency of MN of 36.4 ± 3.5 per 1000 BN cells (control 3.4 ± 0.3). Neither VRB nor VCR was able to induce a clear dose-dependent increase in MN frequency.
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Both chemicals inhibited cell proliferation, which is expressed as a decrease in the percentage of BN cells compared with their respective controls (Table I
). The number of BN cells in VRB-treated lymphocytes was significantly reduced (P < 0.02) to 30.0–34.5% at all doses assayed, while in VCR-treated lymphocytes an important decrease in the percentage of BN cells was observed, ranging from 35.4% at 0.10 µg/ml to 7.6% at 1.00 µg/ml (P < 0.01).
Table II shows the frequencies of centromere-positive (cen+) and centromere-negative (cen–) MN induced by VRB and VCR. In controls, 30/111 (27.03%) MN analyzed showed cen+ and 81/111 (72.97%) MN were cen–. After treatment with 0.01 µg/ml VRB or VCR, 211/235 (89.79%) and 113/129 (87.60%) MN showed cen+, respectively. The positive control COL at 0.01 µg/ml exhibed similar values (90.73%). The percentage of positive signals was statistically significant in treated cultures when compared with that of the control (P < 0.001).
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Discussion |
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VRB is a semisynthetic vinca alkaloid that differs chemically from the naturally occurring compound VCR in the configuration of the catharanthine ring structure (Potier, 1989
). This structural modification may be responsable for its lower toxicity and its increased antitumor activity compared with the analog (Krikorian et al., 1989).
To evaluate MN induction in human lymphocyte cultures, the MN assay in cytokinesis-blocked lymphocytes proved to be a suitable method for detecting spindle disturbances and errors in chromosome segregation (Migliore and Nieri, 1991).
Both VRB and VCR were strong inducers of micronucleated lymphocytes. The findings on induction of MN by VCR are in agreement with numerous previous reports, both in vitro (Migliore and Nieri, 1991; Surrallés et al., 1994; Sgura et al., 1997) and in vivo (Yamamoto and Kikuchi, 1980; Tinwell and Ashby, 1991; Kondo et al., 1992). No previous MN studies have been done with VRB.
A comparison of the results obtained with both compounds suggests that VCR is a more potent aneugenic agent than VRB. At a dose of 0.50 µg/ml, VRB produced an ~5-fold increase with respect to the control frequency of BNMN, while with VCR, this frequency increased 10-fold.
We have not observed any clear dose-dependent effect with either chemical tested. The decrease in the frequency of MN at the highest dose would suggest mitotic delay and/or cell death. A delay in cellular proliferation was evidenced by a decrease in the percentage of BN cells. Farooqi et al. (1993) reported the same phenomenon with the aneuploidy-inducing agents VCR, vinblastine and COL in binucleated splenocytes. In our previous work we found that VRB at 1.0 µg/ml caused inhibition of human lymphocyte progression (González Cid et al., 1997b)
Futhermore, cells with a high level of damage may tend to be eliminated, possibly by apoptosis, prior to completing the nuclear division process required for MN expression (Fenech et al., 1997).
The identification of centromeres in MN by FISH was performed to determine the ratio between MN produced by lagging whole chromosomes and those resulting from acentric chromosomal fragments. FISH experiments in human lymphocytes with a centromere-specific -satellite DNA probe, common for all chromosomes, showed that 89.79% of VRB-induced MN and 87.60% of VCR-induced MN were cen+, indicating that they contained whole chromosomes. These results are comparable with those previously published with different spindle poisons. Eastmond and Tucker (1989) showed that micronucleated lymphocytes induced by COL and VCR contained 92% and 87% kinetochore-positive MN, respectively. Farooqi et al. (1993) found that treatment of mouse splenocytes in vitro with COL, VCR and vinblastine increased the percentage of cen+ MN, to between 83 and 91%. Similarly, Huber et al. (1996) reported that vinblastine induced 72–89% cen+ MN in human lymphocytes using an -satellite pancentromeric DNA probe.
If all MN inducing agents are pure aneugens we would expect to find that these agents induce 100% cen+ MN. However, after treatment with VRB and VCR at the doses tested, 10–12% of the MN lack centromeric signals. Grawé et al. (1994) found that neither VCR nor COL induce 100% cen+ MN, indicating that these compounds also have a clastogenic effect. Migliore et al. (1996) reported that cen– MN can arise from spontaneous chromosomal aberrations. It is also possible that some cen– MN are due to failed hybridization.
VRB and VCR interact with the microtubular proteins, which results in an abnormal distribution of chromosomes, causing aneuploidy. The present study confirmed the mode of action of both vinca alkaloids, VRB having a lower aneugenic potencial. Our results suggest that treatment with VRB should lead to less genetic risk for patients receiving this drug.
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Acknowledgments |
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This work was supported by grants from the National Research Council (CONICET) and Alberto J.Roemmers Foundation.
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Notes |
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1 To whom correspondence should be addressed. Tel: +54 1 805 8803; Fax: +54 1 803 9475; Email: postmaster{at}anmra.sld.ar
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Received on January 28, 1998; accepted on June 12, 1998.
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